Water-only Toxicity Tests Research Articles

Novel Reproduction Toxicity Test Method for Hyalella azteca Using Sexually Mature Amphipods Provides More Robust Data Than Standard Methods in Exposures to Imidacloprid.

Hyalella azteca is an epibenthic crustacean used in ecotoxicology, but there are challenges associated with standard methods using reproduction as an endpoint. A novel, 28-day reproduction toxicity test method for H. azteca was created to address these issues by initiating tests with sexually mature amphipods to eliminate the confounding effects of growth, using a sex ratio of seven females to three males to reduce reproductive variability, and conducting tests in water-only conditions to make recovery of juveniles easier and expand testing capabilities to water-soluble compounds. In the present study, we evaluated the 28-day novel method by comparing it with the 42-day standard test method in duplicate and parallel water-only, static-renewal exposures to sublethal concentrations of imidacloprid (0.5-8 µg/L). Both methods showed similar effects on survival, with survival approaching 50% in the highest test concentration (8 µg/L). However, the 42-day median effect concentrations (EC50s) for growth were more sensitive in the standard method (1.5-3.2 µg/L) compared with the 28-day EC50s generated by the novel method (>8 µg/L). Reproduction endpoints (juveniles/female) produced similar EC50s between methods, but the data were less variable in novel tests (smaller coefficients of variation); therefore, fewer replicates would be required to detect effects on reproduction compared with the standard method. In addition, novel tests generated 28 days of reproduction data compared with 14 days using standard tests and allowed survival and growth of sexes to be assessed independently. Thus, the novel method shows promise to improve the use of reproduction as an endpoint in water-only toxicity tests with H. azteca. Environ Toxicol Chem 2024;43:723-735. © 2024 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Toxicity assessment of typical polycyclic aromatic hydrocarbons to Daphnia magna and Hyalella azteca in water-only and sediment–water exposure systems

While the equilibrium partitioning (EqP) method has been demonstrated to effectively predict the adverse effects of nonionic organic chemicals in sediment on benthic organisms by sediment toxicity tests, only a limited number of studies have been performed both in water-only and whole-sediment toxicity tests using the same species and verified the validity of EqP-based toxicity assessment. To further examine the validity of the EqP method for application in a wide range of hydrophobicity, we conducted sorption/desorption experiments and both water-only and sediment toxicity tests using a popular aquatic crustacean species, Daphnia magna (48 h), and benthic species Hyalella azteca (96 h) for six typical polycyclic aromatic hydrocarbons (PAHs) with three to five rings and an amine derivative: anthracene, phenanthrene, fluoranthene, pyrene, benzo[a]pyrene, dibenzo[a,h]anthracene, and 1-aminopyrene. The linear sorption coefficient was determined and ranged from 2.7 × 102 (phenanthrene) and 1.2 × 104 L/kg (benzo[a]pyrene) highly depending on the hydrophobicity while the aqueous concentrations were stable after 24 h in the desorption test. As result of acute toxicity tests in the water-only exposure system, anthracene and dibenz[a,h]anthracene were found to be nontoxic to both species, while median effect/lethal concentrations (EC50/LC50) were determined as ranging from 0.66 (benzo[a]pyrene) to 330 μg/L (phenanthrene), and from 11 (1-aminopyrene) to 180 μg/L (phenanthrene) for D. magna and H. azteca, respectively. Among these compounds, three PAHs with three, four, and five rings each, and 1-aminopyrene were subjected to sediment-water toxicity tests. In the sediment-water tests, the LC50 of phenanthrene and pyrene was three to six times higher than that of the water-only tests for H. azteca while the EC50 was 1.1 to 2.0 times higher for D. magna. In contrast, the EC50/LC50 of benzo[a]pyrene (BaP) in the sediment-water toxicity test was more than 5 times higher than that in the water-only test for both H. azteca and D. magna. The EC50/LC50 values of 1-aminopyrene were similar in both the sediment-water and the water-only toxicity tests, ranging narrowly from 21 to 28 μg/L and 8.8 to 11 μg/L for D. magna and H. azteca, respectively. The EC50/LC50 based on the body residue (ER50/LR50) was investigated for two of the representative PAHs, pyrene, and BaP. The ER50/LR50 of pyrene in both species was 2.3 and 11 times higher in the water-only toxicity test for D. magna and H. azteca, respectively, while those of BaP in the sediment-water toxicity test were not calculated for the sediment-water toxicity tests, and the highest body concentration in the sediment-water tests was lower than the ER50/LR50 in the water-only toxicity test. Although the experimental results were comparable with the predicted sediment toxicity values based on the EqP method for the selected PAHs in this study, there is a risk of phenanthrene and pyrene being slightly underestimated (1.4–1.9 fold for phenanthrene and 3.7–6.1 fold for pyrene) by the EqP method for H. azteca. These results reaffirm that the bioavailability of poorly water-soluble chemicals is important for sediment toxicity and that the exposure pathway should be further investigated to avoid under- and overestimation via the EqP method.

Chironomus sancticaroli (Diptera, Chironomidae) as a Sensitive Tropical Test Species in Laboratory Bioassays Evaluating Metals (Copper and Cadmium) and Field Testing.

Despite that chironomids are the most widely used benthic insect test species worldwide, little research has been conducted so far with tropical chironomid representatives. This study was designed to evaluate the indigenous midge Chironomus sancticaroli as a candidate test species for use in tropical toxicity assessments. To this end, laboratory water-only toxicity tests were conducted evaluating copper and cadmium. Obtained lethal concentration values were overall comparable or lower than those reported for other chironomids, including those most commonly used in temperate regions (C. riparius and C. dilutus). In addition, C. sancticaroli was deployed in situ in the Monjolinho River (São Paulo State, Brazil), and toxicity of sediment from this river was evaluated in the laboratory. Several field water and sediment quality parameters also were measured to enable correlating these with the effects observed in these toxicity tests. Field sediment toxicity to C. sancticaroli appeared to be related with sediment endosulfan concentrations, whereas effects noted in the in situ test were likely due to low pH values measured in river water. Chironomus sancticaroli appears to be a suitable candidate for inclusion as a test species in tropical toxicity evaluations in both the laboratory and the field.

Comparative sensitivity of field and laboratory populations of Hyalella azteca to the pyrethroid insecticides bifenthrin and cypermethrin

Hyalella azteca are epibenthic invertebrates that are widely used for toxicity studies. They are reported to be more sensitive to pyrethroid insecticides than most other test species, which has prompted considerable use of this species in toxicity testing of ambient surface waters where the presence of pyrethroids is suspected. However, resident H. azteca have been found in some ambient water bodies reported to contain surface water and/or sediment pyrethroid concentrations that are toxic to laboratory reared H. azteca. This observation suggests differences in the sensitivities of laboratory reared and field populations of H. azteca to pyrethroids. The goal of the present study was to determine the sensitivities of laboratory reared and field populations of H. azteca to the pyrethroids bifenthrin and cypermethrin. Specimens of H. azteca were collected from resident populations at field sites that are subject to varied land-use activities as well as from laboratory populations. These organisms were exposed to bifenthrin- or cypermethrin-spiked water in 96-h water-only toxicity tests. The resulting data demonstrated that: 1) field-collected populations in urban and agricultural settings can be >2 orders of magnitude less sensitive to the pyrethroids than laboratory reared organisms; 2) field-collected organisms varied in their sensitivity (possibly based on land-use activities), with organisms collected from undeveloped sites exhibiting sensitivities similar to laboratory reared organisms; and 3) the sensitivity of field-collected "tolerant" organisms increased in subsequent generations reared under laboratory conditions. Potential mechanisms for these differences are discussed.

Effects of triclosan on marine benthic and epibenthic organisms

Triclosan is an antimicrobial compound that has been widely used in consumer products such as toothpaste, deodorant, and shampoo. Because of its widespread use, triclosan has been detected in various environmental media, including wastewater, sewage sludge, surface waters, and sediments. Triclosan is acutely toxic to numerous aquatic organisms, but very few studies have been performed on estuarine and marine benthic organisms. For whole sediment toxicity tests, the sediment-dwelling estuarine amphipod, Ampelisca abdita, and the epibenthic mysid shrimp, Americamysis bahia, are commonly used organisms. In the present study, median lethal concentration values (LC50) were obtained for both of these organisms using water-only and whole sediment exposures. Acute 96-h water-only toxicity tests resulted in LC50 values of 73.4 and 74.3 µg/L for the amphipod and mysid, respectively. For the 7-d whole sediment toxicity test, LC50 values were 303 and 257 mg/kg (dry wt) for the amphipod and mysid, respectively. Using equilibrium partitioning theory, these whole sediment values are equivalent to interstitial water LC50 values of 230 and 190 µg/L for the amphipod and mysid, respectively, which are within a threefold difference of the observed 96-h LC50 water-only values. Triclosan was found to accumulate in polychaete tissue in a 28-d bioaccumulation study with a biota-sediment accumulation factor of 0.23 kg organic carbon/kg lipid. These data provide some of the first toxicity data for triclosan with marine benthic and epibenthic species while also indicating a need to better understand the effects of other forms of sediment carbon, triclosan ionization, and organism metabolism of triclosan on the chemical's behavior and toxicity in the aquatic environment.

ASSESSING THE BIOLOGICAL RELEVANCE OF EXPOSING FRESHWATER ORGANISMS TO ATRAZINE AND MOLINATE IN ENVIRONMENTALLY REALISTIC EXPOSURE TEST SYSTEMS

Assessing the toxicity of chemicals in treated laboratory water may not accurately represent the toxicity of chemicals in natural aquatic systems. In natural water, dissolved organic matter, suspended particulate matter, and sediment play key roles in the sorption of contaminants from the water. Our previously published series of papers illustrated that the presence of sediment in aquatic toxicity testing systems significantly (p < 0.05) reduced the bioavailability of the herbicides atrazine and molinate to five Australian freshwater organisms. It is not clear whether the reduced bioavailability means that the trigger values (TVs) in the current Australian and New Zealand water quality guidelines, which are calculated using toxicity data from water-only toxicity tests, provide appropriate environmental protection. Several new sets of TVs were derived in the present study and were compared to each other and to the current Australian and New Zealand TVs for atrazine and molinate. The current Australian and New Zealand TVs for atrazine and molinate provided appropriate protection to Australian freshwater species. Australian freshwater species have a sensitivity distribution similar to those of overseas species to atrazine and molinate.

Solid phase microextraction fibers for estimating the toxicity of nitroaromatic compounds

Solid phase microextraction fibers are useful for investigating bioavailable organic contaminants in complex environmental matrixes such as aquatic sediments. Solid phase microextraction fibers are polymer-coated silica fibers that sorb dissolved organic compounds from water and sediment. We investigated their concentrations as measures of exposure for 2,4,6-trinitrotoluene and its degradation products in acute sediment and water-only toxicity tests with Tubifex tubifex, Chironomus tentans, and Ceriodaphnia dubia. Results from these exposures allowed us to compare solid phase microextraction fibers concentrations to two conventional measures of toxicant dose: external matrix (water, sediment) and internal (organism) concentrations. Because trinitrotoluene degrades within sediment and organisms, doses based on sediment, water, and organism concentrations were calculated using the molar sum of trinitrotoluene and its nitroaromatic degradation products. Among species and matrixes, median lethal doses based on solid phase microextraction fiber and organism concentrations ranged from 12.6 to 55.3 (μmol nitroaromatic per ml polyacrylate and 83.4 to 172.3 nmol nitroaromatic per gram tissue, wet weight, respectively. In contrast to matrix concentrations, which are specific to sediment or water, both organism and solid phase microextraction fiber concentrations appeared to provide measures of dose independent of exposure scenario (sediment or water). Median lethal doses based on fiber concentrations in whole-sediment and water-only Tubifex tubifex toxicity tests were within a factor of 1.1 (18.7 and 21.3 (μmol nitroaromatic per ml polyacrylate, respectively). Median lethal doses based on organism concentrations were within a factor 1.4 for Chironomus tentans exposed in water-only or whole-sediment scenarios (118.0 and 83.4 nmol nitroaromatic per gram tissue, wet weight, respectively). Solid phase microextraction fibers may provide a powerful chemical estimate of exposure with which to understand bioavailability and toxicity of organic compounds to benthic organisms.

Inheritance of mercury tolerance in the aquatic oligochaete Tubifex tubifex.

Resistance to contaminants is an important yet unmeasured factor in sediment toxicity tests. The rate at which mercury resistance develops and its genetic persistence in the oligochaete worm Tubifex tubifex were studied under laboratory conditions. Worms were raised for four generations under two different sediment treatments, one reference clean sediment, the other contaminated with mercury. Worms raised in mercury-contaminated sediment developed mercury tolerance that persisted even when the worms were raised for three subsequent generations in clean sediment. Mercury tolerance was determined by comparative water-only toxicity tests with mercury as the only stressor. Control worms had a mean lethal concentration (LC50) of 0.18 mg/L(-1). Worms exposed to high levels of mercury in sediment had high mercury tolerance with a mean LC50 of 1.40 mg/L(-1). When mercury-tolerant and control mercury-intolerant worms were crossed, their descendants also demonstrated mercury tolerance during lethal toxicity tests. The LC50 for worm descendants resulting from this cross was 1.39 mg/L(-1). Adaptation to mercury exposures occurred rapidly in this group of worms and appears to be due to both phenotypic and genotypic mechanisms. Development of contaminant resistance and adaptation may be common phenomena in aquatic benthic invertebrates, which should be considered during the design and interpretation of toxicity tests.

Sensitivity of the amphipod Tiburonella viscana (Platyischnopidae) to K2CR2O7

The sensitivity of Tiburonella viscana to K2Cr2O7 was evaluated from 18 water-only toxicity tests. The mean 48h-LC50 was estimated as 11.217 ± 3.768 mg/l, and the warning limits were calculated (mean LC50 ± 2 SD) as being between 3.68 and 18.75 mg/l. These results are baseline for the species and can be used for future comparisons in toxicity tests.

An assessment of the toxicity of phthalate esters to freshwater benthos. 2. Sediment exposures

Seven phthalate esters were evaluated for their 10-d toxicity to the freshwater invertebrates Hyalella azteca and Chironomus tentans in sediment. The esters were diethyl phthalate (DEP), di-n-butyl phthalate (DBP), di-n-hexyl phthalate (DHP), di-(2-ethylhexyl) phthalate (DEHP), diisononyl phthalate (DINP), diisodecyl phthalate (DIDP), and a commercial mixture of C7, C9, and C11 isophthalate esters (711P). All seven esters were tested in a sediment containing 4.80% total organic carbon (TOC), and DBP alone was tested in two additional sediments with 2.45 and 14.1% TOC. Sediment spiking concentrations for DEP and DBP were based on LC50 (lethal concentration for 50% of the population) values from water-only toxicity tests, sediment organic carbon concentration, and equilibrium partitioning (EqP) theory. The five higher molecular weight phthalate esters (DHP, DEHP, DINP, DIDP, 711P), two of which were tested and found to be nontoxic in water-only tests (i.e., DHP and DEHP), were tested at single concentrations between 2,100 and 3,200 mg/kg dry weight. Preliminary spiking studies were performed to assess phthalate ester stability under test conditions. The five higher molecular weight phthalate esters in sediment had no effect on survival or growth of either C. tentans or H. azteca, consistent with predictions based on water-only tests and EqP theory. The 10-d LC50 values for DBP and H. azteca were >17,400, >29,500, and >71,900 mg/kg dry weight for the low, medium, and high TOC sediments, respectively. These values are more than 30x greater than predicted by EqP theory and may reflect the fact that H. azteca is an epibenthic species and not an obligative burrower. The 10-d LC50 values for DBP and C. tentans were 826, 1,664, and 4.730 mg/kg dry weight for the low, medium, and high TOC sediments, respectively. These values are within a factor of two of the values predicted by EqP theory. Pore-water 10-d LC50 values for DBP (dissolved fraction) and C. tentans in the three sediments were 0.65, 0.89, and 0.66 of the water-only LC50 value of 2.64 mg/L, thereby agreeing with EqP theory predictions to within a factor of 1.5. The LC50 value for DEP and C. tentans was >3,100 mg/kg dry weight, which is approximately 10x that predicted by EqP theory. It is postulated that test chemical loss and reduced organism exposure to pore water may have accounted for the observed discrepancies with EqP calculations for DEP

An assessment of the toxicity of phthalate esters to freshwater benthos. 1. Aqueous exposures

Tests were performed with the freshwater invertebrates Hyalella azteca, Chironomus tentans, and Lumbriculus variegatus to determine the acute toxicity of six phthalate esters, including dimethyl phthalate (DMP), diethyl phthalate (DEP), di-n-butyl phthalate (DBP), butylbenzyl phthalate (BBP), di-n-hexyl phthalate (DHP), and di-2-ethylhexyl phthalate (DEHP). It was possible to derive 10-d LC50 (lethal concentration for 50% of the population) values only for the four lower molecular weight esters (DMP, DEP, DBP, and BBP), for which toxicity increased with increasing octanol-water partition coefficient (Kow) and decreasing water solubility. The LC50 values for DMP, DEP, DBP, and BBP were 28.1, 4.21, 0.63, and 0.46 mg/L for H. azteca; 68.2, 31.0, 2.64, and > 1.76 mg/L for C. tentans; and 246, 102, 2.48, and 1.23 mg/L for L. variegatus, respectively. No significant survival reductions were observed when the three species were exposed to either DHP or DEHP at concentrations approximating their water solubilities.

ACCUMULATION AND TOXICOKINETICS OF FLUORANTHENE IN WATER-ONLY EXPOSURES WITH FRESHWATER AMPHIPODS

Two 10-d water-only toxicity tests with radiolabeled fluoranthene were conducted with two species of freshwater amphipods, Hyalella azteca and Diporeia sp. For H. azteca, 10-d median lethal concentrations were 564 nmol/L and 481 nmol/L. Tentative median lethal doses, determined from the regressions of body burden of remaining live H. azteca versus survival, were 5.6 and 3.6 mmol fluoranthene/kg wet weight tissue. Diporeia appeared to be less sensitive, because survival in Diporeia was greater than 84% after 10-d exposures. Elimination rates determined for Diporeia, ranging from 0.0011 to 0.0042/h (half-lives of 7–26 d), were much slower than rates determined for H. azteca of 0.128 to 0.188/h (half-lives of 4–6 h). Faster elimination in H. azteca may be related to its greater ability to metabolize fluoranthene. For H. azteca, an average of 17% of its body burden was present as metabolites after 24 h of exposure to radiolabeled fluoranthene, as compared to 5% for Diporeia. For Diporeia, exposure to various water concentrations of fluoranthene for various lengths of time resulted in declines in the conditional uptake clearance rates (ml water cleared/g wet weight tissue/h). A similar, although less dramatic trend was observed for conditional uptake clearance rates in H. azteca.

Organic carbon partitioning as a basis for predicting the toxicity of chlorpyrifos in sediments

AbstractThe objective of this study was to evaluate an organic carbon partitioning model for predicting bioavailability of the organophosphate chlorpyrifos in sediments, in support of the development of a sediment‐quality criterion for the pesticide. Initial 10‐d water‐only toxicity tests were conducted with the midge Chironomus tentans to define the sensitivity of this species to chlorpyrifos. Two uncontaminated sediments with differing organic carbon contents (approximately 3 and 8.5%) were spiked with varying amounts of chlorpyrifos designed to result in equilibrium interstitial (pore) water concentrations that would bracket the effects concentrations observed in the water‐only exposures. Ten‐day toxicity tests with Chironomus tentans were conducted with the spiked sediments under conditions similar to those in the water‐only exposure. Based on predicted pore‐water concentrations of chlorpyrifos, results of the water‐only and sediment tests were in close agreement. The water‐only LC50 of chlorpyrifos to Chironomus tentans was 70 ng/L, whereas LC50 values based on predicted pore‐water concentrations in the two test sediments were 40 and 70 ng/L. Total concentrations of chlorpyrifos measured in pore water from the test sediments were always at least an order of magnitude greater than predicted pore‐water concentrations of the pesticide. However, upon correction for that fraction of the measured chlorpyrifos potentially bound to DOC in the pore water, measured and predicted chlorpyrifos concentrations were in much closer agreement, with the former typically about threefold greater than the latter. Overall, these results suggest that within the range of organic carbon tested in the present study, an equilibrium partitioning model based on organic carbon is appropriate for predicting the bioavailability of sediment‐associated chlorpyrifos to benthic invertebrates.